1、藥物代謝及其動(dòng)力學(xué)在新藥研發(fā)中的應(yīng)用,胡卓漢 博士 瑞德肝臟疾病研究(上海)有限公司 復(fù)旦大學(xué)藥學(xué)院,2004年12月30日 中國.北京,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),藥物研發(fā)的三大任務(wù) 藥效 Efficacy/ Pharmacodynamics 安全 Safety / Toxicology 藥物代謝動(dòng)力學(xué) Drug Metabolism/Pharmcokinetics,藥物代謝動(dòng)力學(xué)的任務(wù)

2、,（最大無毒性濃度）,(最小有效濃度）,(最小藥效時(shí)間）,血漿濃度,時(shí)間,藥效,毒理,藥代,最佳 血漿濃度,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段 能否被吸收？ permeability 是否被代謝？ metabolic stability 代謝產(chǎn)物？ metabolite identification 代謝途徑？ pathway identification 對(duì)其它藥物的影響？ dr

3、ug-drug interaction,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),臨床前階段 生物利用度 bioavailability 血漿濃度的線性和非線性 dose escalation & proportionality 多次給藥和體內(nèi)積蓄 multiple doses & accumulation 吸收和排泄模式 mass balance 體內(nèi)分布 distribution 從動(dòng)物代謝推算人體

4、代謝 extrapolation,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),臨床階段 長期毒性實(shí)驗(yàn)的動(dòng)物選擇 metabolism profiling in animals and humans,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實(shí)驗(yàn)

5、,臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),臨床實(shí)驗(yàn)準(zhǔn)則 Good Clinical Practice (GCP),非臨床實(shí)驗(yàn)準(zhǔn)則 Good Laboratory Practice (GLP),二五原則 5 毫克 5 天,臨床前實(shí)驗(yàn)藥物代謝動(dòng)力學(xué)的生物模型 體外和離體模型 (in vitro / in situ models) 吸收模型 absorption/permeability 代謝模型 metabolism 體外推測(cè)和體內(nèi) (in vitro / in vivo correlation) 動(dòng)物模型 (in vivo animal models) 動(dòng)物推測(cè)人 (species extrapolation)

6、,排出太快/藥效時(shí)間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,Situation Analysis,in vitro體外 metabolism,in situ離體 permeability,in vivo體內(nèi) bioavailability,Plasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orally,Poor oral bioavaila

7、bility,藥物吸收模型,計(jì)算機(jī),脂溶度,脂層轉(zhuǎn)移,細(xì)胞層轉(zhuǎn)移,十二指腸灌流,14,absorption/distribution model 脂層轉(zhuǎn)移模型,水相 Aqueous phase,水相 Aqueous phase,有機(jī)相 Organic phase,pH=6.5,pH=7.4,Permeability Evaluation in vitro,15,in vitro absorption/distribution model,Caco-2 Transport Pathways人大腸癌細(xì)胞模型,Transport Pathways藥物吸收機(jī)制,被動(dòng),細(xì)胞間,主動(dòng),P糖蛋白,Probe

8、s for Transport Pathways腸道吸收標(biāo)準(zhǔn)對(duì)照藥物,Transcellular （被動(dòng)吸收） Propranolol, Testosterone Paracellular （細(xì)胞間滲透） Mannitol, Inulin Carrier mediated （主動(dòng)吸收） Glucose P-Glycoprotein mediated （P糖蛋白調(diào)節(jié)） 底物 Vinblastine抑制物 Verapamil,Glucose （蔗糖） vs Inulin （木香素）主動(dòng)吸收 vs 細(xì)胞間滲透,Propranolol vs Mannitol被動(dòng)吸收 vs 細(xì)胞間滲透,由P蛋白所調(diào)節(jié)的

9、藥物吸收使用P糖蛋白抑制劑 Verapamil,Chong, Dando Pharm. Res. 1997,False Positive 假陽性 低,False Negative 假陰性 高,Caco-2 Transport Pathways 人大腸癌細(xì)胞吸收模型,in situ rat intestinal perfusion (single pass) 離體大鼠十二指腸灌流模型（單循環(huán)）,METHOD Animal: Male Sprague-Dawley rats (250 - 350 g), fasted overnight. Rat is anesthetized by uretha

10、ne 1.5g/kg, im. before perfusion starts. Perfusate: Phosphate buffer, pH = 6.5 10 mM glucose Phenol red (negative control) Acetaminophen (positive control) Final concentrations of test article = 0.05-0.30 mg/mL,Perfusion Procedures: rat is put on a heating pad to maintain body temperature jejunum is

11、 exposed via a middle line incision sutures: 1st is made at 5 cm distal to the ligament of Treitz 2nd is made at about 20 cm distal to 1st one the inlet of cannula - a syringe infusion pump the outlet of cannula - a fraction collector the perfusion segment is precleaned by passing 10 ml of blank per

12、fusate buffer perfusion time and rate = 0.1 ml/min for 120 min outlet perfusion samples are collected every 10 min plasma samples are collected at 30, 60, 90 and 120 min after perfusion Calculations: Permeability (Peff, cm/min) = (Q/2RLp) x (1- Cout / Cin ) Cout / Cin = (Cout / Cin) x phenol red in

13、/ phenol red out,in situ rat intestinal perfusion (single pass),In situ rat intestinal permeability (single pass),Prediction within 90% interval = 19/31 (61.3%),In-house validation,假陽性,假陰性,Plasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed 50 mg/kg orally,Poor oral bioavai

14、lability,排出太快/藥效時(shí)間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,Situation Analysis,in vitro體外 metabolism,in situ離體 permeability,in vivo體內(nèi) bioavailability,In Situ Rat Intestinal Permeability: Good,陽性對(duì)照,陰性對(duì)照,受試藥物,Enhanced Throughput Screening Perfusion: 4 compo

15、unds per day (4 animals) Sample size: time points 7 duplicate x 2 control/drug x 3 sample/perfusion 42 Total samples/day168 Bioanalysis: no extraction no standard curve (peak area) machine time/2 LCs24 hrs Total manpower:animal tech x 1 PKDM tech x 2 Test article amount:1 mg / test article Screening

16、 rate:one chemotypes with 30 compounds / 2 weeks,pKa = 10 pKa = 8.4 pKa = 6.5 Preduced%= 0% Preduced%= 7% Preduced%= 12%,SAR: pKa vs. permeability 實(shí)例：結(jié)構(gòu)優(yōu)化和吸收率分析,SAR: permeability vs. efficacy 實(shí)例：結(jié)構(gòu)優(yōu)化和吸收率和活性的分析,IC50 = 2 uM Preduced%= 0%,IC50 = 0.012 uM Preduced%= 0%,IC50 = 1.1 uM Preduced%= 17%,IC50

17、= 0.025 uM Preduced%= 15%,小結(jié)：體外和離體藥物吸收實(shí)驗(yàn)系統(tǒng) 體外人大腸癌細(xì)胞模型 (in vitro Caco-2 monolayer) 離體大鼠十二指腸灌流模型 (in situ rat intestine perfusion) 體內(nèi)動(dòng)物藥物代謝動(dòng)力學(xué)模型 二五原則: 5毫克/5天,血漿濃度,時(shí)間,化學(xué)藥物,化學(xué)藥物+中藥,中藥的藥物代謝動(dòng)力學(xué)的任務(wù) 本身的藥物代謝動(dòng)力學(xué)問題 對(duì)其它藥物吸收的作用,排出太快/藥效時(shí)間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研

18、發(fā)階段,后期研發(fā)階段,Situation Analysis,in vitro體外 metabolism,in situ離體 permeability,in vivo體內(nèi) bioavailability,死還是不死,這是個(gè)問題. To be or not to be, this is a problem. - 哈默雷特 體內(nèi)試驗(yàn)還是體外試驗(yàn), 這是個(gè)問題. In vitro or in vivo, this is a problem. -藥代研究員,動(dòng)物體內(nèi)模型 - 人體內(nèi)(臨床試驗(yàn)) In vivo animals vs. in vivo humans 人體外模型 -人體內(nèi)(臨床試驗(yàn)) In

19、vitro humans vs. in vivo humans 選擇的指南 與人相似：疾病模型，藥效，毒性，藥物代謝 實(shí)驗(yàn)成本,38,Heartbeat and Bodyweight （心率和體重）,小鼠,大鼠,兔,猴,狗,人,39,Liver weight and Hepatic Flow vs Bodyweight （體重，肝重和肝血流量）,人,狗,猴,兔,大鼠,小鼠,人,狗,猴,兔,大鼠,小鼠,40,Antipyrine clearance (l/min),rat,mouse,rabbit,monkey,dog,human,Clearance,In Vitro Models of the

20、 Liver體外肝模型,Hepatocytes 肝細(xì)胞 Liver slices 肝切片 Liver microsomes 肝微粒體 Liver S-9 Fraction 肝S-9組分,USFDA Guidance for Industry美國藥物和食品管理局關(guān)于藥物代謝實(shí)驗(yàn)的指南,“The most complete picture for hepatic metabolism can be obtained with liver systems,in which the cofactors are self-sufficient and the natural orientation fo

21、r linked enzymes is preserved. Isolated hepatocytes and precision-cut slices have these desirable features.”,Guidance for Industry, Drug Metabolism/Drug InteractionStudies in the Drug Development Process: Studies In VitroCDER, CBER, U.S. FDA, 1997,譯文： 肝系統(tǒng)（分離的肝細(xì)胞和精確的肝切片）能為藥物代謝 實(shí)驗(yàn)提供最完全的信息，因?yàn)檫@個(gè)系統(tǒng)含有足夠的天

22、然 水平的酶系。,2-Hydroxy-EE2,Conjugates,EE2,EE2,Hepatocytes （肝細(xì)胞）,Microsomes（微粒體） Hepatocytes（肝細(xì)胞）,Metabolism of Eythinyl Estradiol (EE2) 肝微粒體和肝細(xì)胞的代謝功能差異,Li, Hartman, Lu, Collins and Strong, Br J Clin Pharmacol 48, 733-742(1999),Plasma concentrations of BCH-3840 and its metabolite (BCH-6440) in mice dosed

23、 50 mg/kg orally,Poor oral bioavailability,排出太快/藥效時(shí)間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,Situation Analysis,in vitro體外 metabolism,in situ離體 permeability,in vivo體內(nèi) bioavailability,Reaction volume:1.0 ml, DPBS pH 7.4 Hepatic S-9/Microsomes:0.5 mg proti

24、en/mL Species:Human/Monkey/Dog/Rat/Mouse Substrate concentration:10 mM NADPH:2.4 mM UDPGA:1.5 mM Incubation:60 min at 37oC Stopping procedure:chilled acetonitrile, 3 x volume,In Vitro Metabolism Assay 體外肝微粒體實(shí)驗(yàn),1 2 3 4,A B C D E F,Enhanced Throughput Screening （增速篩選）,A-B: （空白對(duì)照）：test article + buffer

25、 = vehicle control (VC) C-D:（陰性對(duì)照）：test article + microsomes = negative control (NC) E-F: （實(shí)驗(yàn)樣品）：test article + microsomes + cofactors = treated Dosing solution = time zero (T = 0) 4 compounds including positive reference* / plate * 7 ethoxycoumarin,陰性對(duì)照,空白對(duì)照,測(cè)試樣本,Enhanced Throughput Screening Incub

26、ation: 4 compounds per 24-well plate 15 compounds + 1 positive control per day Sample size: Time zeroduplicate (16 x 2) VCduplicate (16 x 2) NCduplicate (16 x 2) Treatedduplicate (16 x 2) Total samples/day 128 Bioanalysis: no extraction no standard curve (peak area) machine time/2 LCs24 hrs Total ma

27、npower:PKDM tech x 3 Test article amount:0.1 mg / test article Screening rate:one chemotype with 60 compounds / 1 week,HPLC profiles of BCH-3840 and its metabolite (BCH-6440),BCH-3840,metabolite?,In vitro metabolic stability by rat hepatic S9,Efficacy Hits,Optimized Lead Go or no go decision,Compoun

28、d for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段 能否被吸收？ permeability 是否被代謝？ metabolic stability 代謝產(chǎn)物？ metabolite identification 代謝途徑？ pathway identification 對(duì)其它藥物的影響？ drug-drug interaction,Liquid Chromatography / Mass Spectrum of BCH-3840 and its metabolite (BCH-6440),Hydroxylati

29、on or Oxidation,MH+ = 310,MH+ = 294,Mass Identification,HPLC profiles of BCH-3840 and its metabolite (BCH-6440),Preparation of metabolite by bulk incubation,M,M,P,P,10 mg microsomal protein 2 mg BCH-3840,Fraction collection of metabolite,fractionation,concentration,Nuclear Magnetic Resonance profile

30、s of BCH-3840 and its metabolite (BCH-6440),C5-H,BCH-3840,Metabolite,Structure Elucidation,In vitro therapeutic index of BCH-6440,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段 能否被吸收？ permeability 是否被代謝？ metabolic stabili

31、ty 代謝產(chǎn)物？ metabolite identification 代謝途徑？ pathway identification 對(duì)其它藥物的影響？ drug-drug interaction,Inhibitors for CYP IsoformConc (mM) Furafulline (CYP1A2) 10 Tranylcypromine (CYP2A6) 50 Sulfaphenazole (CYP2C9) 25 Omeprazole (CYP2C19) 20 Quinidine (CYP2D6) 2 4-methylpyrazole (CYP2E1) 250 Ketoconazole (

32、CYP3A4) 5,Chemical Inhibition （化學(xué)抑制）,Pure enzyme （純酶） Correlation Analysis （相關(guān)分析）,Metabolism Phenotyping 代謝途徑鑒定,Inhibitors for CYP IsoformConc (mM) Inhibition (% of NC) Tranylcypromine (CYP2A6) 5040.2 Sulfaphenazole (CYP2C9) 2514.2 4-methylpyrazole (CYP2E1) 25067.6 Ketoconazole (CYP3A4) 575.2,Metabo

33、lism Phenotyping 代謝途徑鑒定,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),研究和發(fā)現(xiàn)階段 能否被吸收？ permeability 是否被代謝？ metabolic stability 代謝產(chǎn)物？ metabolite identification 代謝途徑？ pathway identification 對(duì)其它藥物的影響？ drug-drug interaction,Drug-Drug

34、Interactions （對(duì)其它藥物代謝的影響） Inhibition （抑制） potential - IC50 and Ki mechanism - mechanistic（機(jī)械性） competitive （競爭性） test system: liver microsomes （肝微粒體） cryopreserved hepatocytes （冷凍肝細(xì)胞） Induction（誘導(dǎo)） test system: fresh isolated hepatocytes （肝細(xì)胞） Target Enzymes Cytochrome P450s: 1A2, 2A6, 2C8, 2C9, 2C1

35、9, 2D6, 2E1, 3A4 Phase II conjugation: glucuronidation,IC50 (M):0.675 Goodness of Fit:0.9807 95% Confidence Intervals:5.638.28,IC50 (M):20.4 Goodness of Fit:0.9730 95% Confidence Intervals:16.9-26.3,CYP3A4,CYP3A4,Drug-drug interaction: inhibition 抑制作用,體外藥效濃度 = 1 uM,Drug-drug interaction: Induction （

36、肝細(xì)胞誘導(dǎo)模型）,5 days procedure Day 0: Isolate fresh hepatocytes, viability 70% Plating hepatocytes to 24-well plate, 0.7 x 106 viable cells/well Plating media replaced with sandwich after 7-hour attachment Day 1:incubation for establishing basal levels of CYP450 isoforms. Day 2:same as Day 1 Day 3:dosing

37、 with test articles Day 4:same as Day 3 Day 5:washing out the dosing solution and adding substrates for CYP450 isoforms as below: 1A2 - ethocyresorufin O-deethylation 2A6 - coumarin 7-hydroxylation 2C9 - tolbutamide 4-hydroxylation 2C19 - S-mephenytoin 4-hydroxylation 2D6 - dextromethorphan O-demeth

38、ylation 2E1 - chlorzoxazone 6-hydroxylation 3A4 - testosterone 6b - hydroxylation,Drug-drug interaction: Induction 誘導(dǎo)作用,排出太快/藥效時(shí)間太短,口服吸收差/血漿濃度太低,分布,排瀉,代謝問題,吸收問題,蛋白質(zhì)相互作用,分布體積,腎臟排泄,肝臟代謝,溶解度,腸道吸收膜通透性,腸道消化,早期研發(fā)階段,后期研發(fā)階段,Situation Analysis,in vitro體外 metabolism,in situ離體 permeability,in vivo體內(nèi) bioavailab

39、ility,Efficacy Hits,Optimized Lead Go or no go decision,Compound for Development (CD),NEW DRUG,IND,NDA,R&D,臨床實(shí)驗(yàn),臨床前實(shí)驗(yàn),研究和發(fā)現(xiàn),臨床前階段 生物利用度 bioavailability 血漿濃度的線性和非線性 dose escalation & proportionality 多次給藥和體內(nèi)積蓄 multiple doses & accumulation 吸收和排泄模式 mass balance 體內(nèi)分布 distribution 從動(dòng)物代謝推算人體代謝 extrapolati

40、on,119%,236%,310%,Proportionality 血漿濃度的非線性,提示： 代謝或排泄的非線性飽和,90%,72%,Proportionality: AUC （大鼠試驗(yàn)）,93%,63%,提示 ：藥物吸收的非線性飽和,TOXICOKINETICS 毒物代謝動(dòng)力學(xué)試驗(yàn) Animal: Sprague-Dawley rats (male & female) Cynomolgus monkey (male & female) Single dose escalation （線性動(dòng)力學(xué)） (50, 250, 500 mg/kg) Multiple dose escalation （藥

41、物體內(nèi)積累） (50, 250, 500 mg/kg, daily for 14 days),90%,72%,Proportionality: AUC （大鼠試驗(yàn)）,93%,63%,提示 ：藥物吸收的非線性飽和,0,100,200,300,400,500,600,0,10,20,30,40,50,60,Female Rats,Oral Dose (mg/kg),0,100,200,300,400,500,600,0,10,20,30,40,50,Male Rats,Oral Dose (mg/kg),Cmax,(,m,g/mL),73%,47%,56%,49%,Proportionality:

42、 Cmax （大鼠試驗(yàn)）,提示 ：藥物吸收的非線性飽和,0.92,0.77,1.04,1.19,1.02,1.07,Accumulation Ratio 藥物積累率 （大鼠）,Male rats,Female rats,Proportionality: AUC （獼猴）,Male Monkey,Female Monkey,49%,34%,60%,38%,提示 ：藥物吸收的非線性飽和,38%,31%,55%,32%,Proportionality: Cmax （獼猴）,Male Monkey,Female Monkey,提示 ：藥物吸收的非線性飽和,Male Monkey,Female Monkey,0.79,1.11,1.12,0.73,0.76,1.14,Accumulation Ratio 藥物積累率 （獼猴）,Phase I Trial (Single dose escalation) 臨床一期單劑量藥代動(dòng)力學(xué)試驗(yàn) Healthy Male Subject (n): 22 Oral Doses (4): 100, 200, 400, and 800 mg Time